In 2002, a total of 518 protein kinase genes in human kinome were identified by Manning et al., in which 218 genes are closely related to the occurrence and development of human diseases (Manning G, et al. 2002, Science, 298:1912-1934). In the drugs obtained up to now, there are as many as 20% of pharmaceuticals using enzymes as the targets, and particularly, the drugs targeting protein kinases are of special value in clinical applications.
Protein kinase is a type of intracellular messenger-dependent enzyme that catalyzes the phosphorylation of particular proteins and implements signaling processes, which mainly includes tyrosine protein kinases (JAKs, Src, Abl, EGFR, FGFR, PDGFR etc.), serine/threonine protein kinases (PKC, MAPK, Rho kinases etc.), bispecific protein kinases (MAPKK) and phosphatidyl inositol kinase (PI3K). The phosphorylation/dephosphorylation process of a protein kinase is able to regulate various biological processes in different cells, such as metabolism, cell differentiation, cell survival, apoptosis, organogenesis, angiogenesis, and immune response etc. (Shchemelinin I., et al. 2006, Folia Biol., 52: 81-100).
JAK kinases (Janus kinases, referred to as JAKs for short, including 4 known members: JAK3, JAK1, TYK2, and JAK2) are a small family in intracytoplasmic non-receptor tyrosine protein kinase superfamily. JAK3 is distributed in marrow and lymphatic system, while JAK1, TYK2, and JAK2 are widely distributed in a plurality of tissue cells. When JAKs bind to a cytokine receptor on the surface of cell, the receptor-coupled JAKs is activated and thereby the receptor is phosphorylated, which provides recruiting response sites, i.e., JAKs phosphorylated STAT proteins, for cytoplasmic signal transducers and activators of transcription, STAT proteins (STAT1˜4, STAT5a, STAT5b, and STAT6). After dimerization, the JAKs phosphorylated STAT proteins are transferred to the nucleus and regulate gene expression. This pathway is called JAK/STAT signaling pathway (O'Shea J. J., et al. 2013, N. Engl. J. Med., 368: 161-170).
The JAK/STAT signaling pathway is a signal transduction pathway stimulated by a plurality of cytokines and growth factor receptors. These factors include interleukins (IL-2˜7, IL-9, IL-10, IL-15, and IL-21), interferons (IFN-α, IFN-β, and IFN-γ), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), growth hormone (GH), prolactin (PRL), thrombopoietin (TPO) etc., which are involved in the proliferation of immune cells and hematopoietic stem cells, and play a key role in immunoregulatory biological processes (Ghoreschi K., et al. 2009, Immunol. Rev., 228: 273-287). Different subtypes of JAK kinases can be activated by diverse receptors, so as to achieve distinct biological functions.
JAK1 can bind to IL-10, IL-19, IL-20, IL-22, IL-26, IL-28, IFN-α, IFN-γ, IL-6 in gp130 family and other γc-containing receptors (Rodig S J., et al. 1998, Cell, 93: 373-383). The knock-off experiment of JAK1 gene in a mouse model has indicated that this enzyme plays a key role in the biological effects of such lots of cytokine receptors described above (Kisseleva T., et al. 2002, Gene, 285: 1-24). JAK1 is a novel target for diseases such as immune-related diseases, inflammation and cancers etc. JAK1 inhibitors can be used for the treatment/prevention of diseases, including but not limited to, autoimmune diseases, inflammation and tumors (Hornakova T., et al. 2010, Blood, 115:3287-3295), such as leukaemia, lymphomata, melanoma, arthritis, psoriasis, Crohn's disease, lupus erythematosus, acquired immunodeficiency syndrome, Behcet's disease (Hou S., et al. 2013, Hum. Genet., 132:1049-1058), etc.
JAK2 was found to have significant role in the regulation processes of a plurality of receptors, including EPO, GH, PRL, IL-3, IFN-γ etc. (Kisseleva T., et al. 2002, Gene, 285: 1-24; Levy D. E., et al. 2002, Nat. Rev. Mol. Cell Biol., 3: 651-662; O'Shea J. J., et al. 2002, Cell, 109 (suppl.): S121-S131). In a mouse model, JAK2 gene knock-off may result in death of anemic animals (Schindler C. et al. 2007, J. Biol. Chem., 282: 20059-20063); While in human, a base mutation JAK2V617F in JAK2 gene is closely related to the occurrence of myeloproliferative diseases, including polycythemia vera (PV), essential thrombocythemia (ET), idiopathic myelofibrosis (IMF), and chronic myelogenous leukemia (CIVIL) etc. (Ghoreschi K., et al. 2009, Immunol. Rev., 228: 273-287). Thus, JAK2 has become an exact target for the treatment/prevention of such diseases.
JAK3 regulates cell signaling via binding to the gamma common chain (γc) in the cytokine-receptor complexes, such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Mutation in either JAK3 or γc may lead to severe combined immunodeficiency (SCID) (Villa A., et al. 1996, Blood, 88: 817-823). Abnormal activity of JAK3 is represented as significant reduction of T-cells and NK cells, and loss of functions of B-cells, which has a strong impact on the normal biological functions of immune system etc. Based on its functional characteristics and special tissue distribution, JAK3 has become a promising pharmaceutical target for immune system-related diseases, and thus its inhibitors will have great clinical value in the treatment/prevention of rheumatoid arthritis (RA), Crohn's disease, systemic lupus erythematosus, multiple sclerosis, type I diabetes, psoriasis, allergic diseases, asthma, chronic obstructive pulmonary disease, leukaemia, lymphoma, organ transplant and other diseases (Papageorgiou A. C., et al. 2004, Trends Pharm. Sci., 2004, 25: 558-562).
TYK2 is the first member in JAK family, and it can be activated by a plurality of receptors, such as interferons (IFNs), IL-10, IL-6, IL-12, IL-23, and IL-27. In mice, the loss of function of TYK2 may lead to deficiency in the signaling pathways of many cytokine receptors, which may further result in virus infection, and decrease antibacterial immune function, and thus increase the possibility of infection in lung (Kisseleva T., et al. 2002, Gene, 285: 1-24). In addition, the research in the group of Lamer A. C. has demonstrated that TYK2 is helpful to suppress the growth and metastasis of breast cancer (Zhang Q., et al. 2011, J. Interferon Cytokine Res., 31:671-677); recently, this group has also reported that TYK2 facilitates obesity regulation by the differentiation of brown adipose tissue (BAT) in mice and human, so that it may protect organisms from obesity, or even reverse it (Derecka M., et al., 2012, Cell Metab., 16:814-824). This might provide a new opportunity for fat patients suffering from cancers.
In November 2012, pan-JAKs inhibitor Xeljanz (Tofacitinib) from Pfizer was approved by FDA for the treatment of RA. In October 2013, the phase III clinical data of Xeljanz for the treatment of psoriasis were disclosed by the company. Compared to the double-blind test of Enbrel (Etanercept), this drug satisfied the requirements for a non-inferiority trial. However, Xeljanz possesses some side-effects, for example, it may result in reduced amount of erythrocytes and leucocytes, and increased cholesterol level. This might be related to its high JAK2 inhibitory activity and low selectivity (Zak M., et al. 2012, J. Med. Chem., 55: 6176-6193). Therefore, it is highly demanded for the research and discovery of selective JAK inhibitors.
There are several selective JAK inhibitors in different clinical phases used for the treatment of immune system-related diseases, such as RA, Crohn's disease, psoriasis, and myelofibrosis, including selective JAK3 inhibitor VX-509, selective JAK1 inhibitor GLPG0634 (Feist E., et al. 2013, Rheumatology, 52:1352-1357) and INCB39110 (http://www.incyte.com/research/pipeline) etc. Besides, some patents have been disclosed for the selective inhibitors with different structure types: 1) selective JAK3 inhibitors, such as pyrrolo[1,2-b]pyridazine (WO2012125887), pyrazolo[3,4-d]pyrimidine (WO2011048082, WO2011134861, WO2012022681), diaminopyrimidines (WO2011029807, WO2012015972), pyrrolo[2,3-b]pyridine (JP2012012332), diamino-pyridinyl-3-formamide (WO2010061971, US20120108566), pyrrolo[2,3-b]pyrazine (WO2011144584, WO2011144585); 2) selective JAK1 inhibitors, such as tricyclic compounds (WO2011086053), substituted pyrazoles and pyrroles (WO2010135650, WO2011112662), anilinophthalazines (WO2012037132). Additionally, patents have also been disclosed for the selective JAK2 inhibitors and selective TYK2 inhibitors, and the inhibitors with both two subtypes (JAK3/1, JAK1/2), which will not be further described herein.
Inducible T-cell kinase (ITK), also referred to as Emt or Tsk, is one of the non-receptor tyrosine kinases in Tec family. ITK is expressed in T-cells, NKT cells and mast cells. This kinase plays a key role in the signaling pathway regulation of T-cell receptor (TCR), CD28, CD2, chemokine receptor CXCR4 and FcεR etc. Secretion of Th2-type cytokines (including IL-4, IL-5, and IL-13 etc.) plays an important part in the regulation of immune inflammation. ITK deficiency has impact on Th2 cell response, and thereby alleviates chronic or late inflammatory reaction (Sahu N., et al. 2009, Curr. Top. Med. Chem., 9: 690-703; Lin T. A., et al. 2004, Biochemistry, 43: 11056-11062). B-cell lymphocyte kinase (BLK) is one of the non-receptor tyrosine kinases in Src family, which is expressed in B lymphocytes, and relates to the growth and differentiation of B lymphocytes. Tight binding between BLK kinase, or phosphatase and corresponding co-receptors has important effect on the signaling pathway regulation of B-cell receptor (BCR), for example, such kinase may influence the apoptosis and formation retardation of BCR (Texido G, et al. 2000, Mol. Cell Biol., 20: 1227-1233). BLK has also important influence on pre-B-cell receptor-mediated NF-κB activation (Saijo K., et al. 2003, Nat. Immunol., 4: 274-279). Recent researches have demonstrated that BLK is related to the pathogenesis of RA, systemic lupus erythematosus and many other autoimmune diseases (Simpfendorfer K R., et al. 2012, Hum. Mol. Genet., 21: 3918-3925; Génin E., et al. 2013, PLoS One, 8: e61044).
TANK-binding kinasel (TBK1), also referred to as NAK (NF-κB activating kinase) or T2K, is a kind of Ser/Thr protein kinase in IKK family. TBK1 is widely expressed in the stomach, colon, lung, thymus and liver of mouse; and also expressed in the lymphoid and nonlymphoid organs of human, including spleen, brain and kidney etc. This kinase has influence on the regulation of immune response to bacteria and virus, and expression of inflammation-related factors, such as IL-6, TNF-α and IFN-β, etc. In the insulin signaling pathway, TBK1 can mediate the phosphorylation of Ser994 in the insulin receptor and the lipid metabolism. These results have indicated that TBK1 plays an important role in various immunobiological and immunopathological mechanisms (Yu T., et al. 2012, Mediators Inflamm., 2012: 979105-979112; Hammaker D., et al. 2012, Rheumatology, 51: 610-618).
Vascular endothelial growth factor receptor (VEGFR) family including 3 members, i.e., VEGFR-1 (Flt1), VEGFR-2 (KDR/Flk1) and VEGFR-3 (Flt4), consists of 7 extracellular regions with immunoglobulin-like structure, a membrane region and a tyrosine kinase region, in which the tyrosine kinase activity is activated via binding between the receptor and the ligand, such as VEGFs A-F and placenta growth factor, further inducing various biological effects in cells, such as important effects on the growth and differentiation of cell (Shibuya M., et al. 2010, Genes Cancer, 1: 1119-1123). Other researches have demonstrated that VEGFR1 is expressed in endotheliocytes, monocytes and macrophages of a RA patient. VEGFA can activate VEGFR1, and result in the proliferation of endotheliocyte and angiopoiesis. VEGFA protein is highly expressed in synovia, lymph, serum and synovial tissue of a RA patient, and the level of VEGFA is positively correlated with RA. VEGFR2 is expressed in the synovial tissue of a RA patient. VEGF A, C, and D can activate VEGFR2 signaling, and enhance the vascular permeability and angiopoiesis. VEGFC can be detected in various types of cells in RA thickened synovial inner layer, especially in perivascular cells and smooth muscle cells. VEGFR3 can be expressed in monocytes, macrophages, some dendritic cells, capillary vessels of normal mammary tissue and neuroendocrine organs. It has been found in some researches that VEGFR3 contributes to the occurrence of autoimmune diseases, such as RA, inflammatory bowel disease, ulcer diseases and Crohn's disease etc., and lymphoangiogenesis-related tumors. However, relevant mechanism has not been completely understood (D'Aura Swanson C, et al. 2009, Nat. Rev. Rheumatol., 5: 317-324; Aoki Y, et al. 2005, J. Natl. Cancer Inst., 97: 2-3).
The protein kinase inhibitors disclosed herein can be used for the treatment and/or prevention of immune system-related diseases, including but not limited to, RA, psoriasis, Crohn's disease, systemic lupus erythematosus, multiple sclerosis, type I diabetes, allergic diseases, chronic obstructive lung disease, asthma, leukaemia, and lymphoma, etc. At the same time, these compounds or a pharmaceutical composition comprising the compounds as the active ingredients will have maximal clinical efficacy for these diseases in the safe therapeutic window.